Method of optically imaging biological tissues by using fluorescence, in particular for defining regions of interest in tissues to be analyzed by tomography

Abstract

The present invention relates to a method of optically imaging at least one biological tissue, in particular to define areas of interest of tissue(s) to be analyzed by tomography. The method according to the invention comprises the following steps: a) introducing at least one fluorescent marker into the tissue(s); b) exciting the marker by incident light and detecting emission bands relating to fluorescence emitted by the marker in response to that excitation; then c) analyzing the fluorescence in these emission bands; and the step b) comprising: sequentially exciting said marker at n different incident excitation wavelengths λ i , said marker being adapted to be excited by at least two of the wavelengths λ i and to emit in response to each wavelength λ i a series S i of m simultaneous emission bands B j having different maximum wavelengths λ′ j that are substantially the same from one series S i to another; and detecting these series S i in order to deduce therefrom an estimate of the three-dimensional location of said marker in the tissue(s) and/or the mean absorption coefficients of the tissue(s) for the excitation wavelengths λ i .

1. A method of optically imaging at least one biological tissue, the method comprising:
 a) introducing at least one fluorescent marker into said at least one tissue; 
 b) exciting said at least one marker by incident light radiations and detecting bands of emission relating to fluorescence emitted by said at least one marker in response to that excitation; and 
 c) analyzing intensities of fluorescence relative to said emission bands, 
 wherein the step b) includes,
 sequentially exciting said at least one marker at n different incident excitation wavelengths λ i , said at least one marker excitable by at least two of these n wavelengths λ i  and to emit in response to each wavelength λ i  a series S i  of m simultaneous emission bands B j  having m different maximum wavelengths λ′ j  that are substantially same from one series S i  to another (where n and m are independent integers equal to or greater than 2 and where i and j respectively vary from 1 to n and from 1 to m), and 
 detecting at least two of the series S i  that each comprise the m bands B j  emitted simultaneously in order to deduce therefrom in the step c) at least one of an estimate of a three-dimensional location of said marker in the at least one tissue and mean absorption coefficients of the at least one tissue for the excitation wavelengths λ i . 
 
 
     
     
       2. An imaging method according to  claim 1 , wherein said at least one marker is based on a fluorophore or a group of fluorophores that is excitable by the incident excitation wavelengths λ i  and that emits the m bands B j  simultaneously in response to each of the incident excitation wavelengths λ i . 
     
     
       3. An imaging method according to  claim 1  or  2 , wherein the step c) also comprises determining one or more emission ratios between the m maximum wavelengths λ′ j  and one or more transmission ratio(s) of said at least one tissue between the n different incident excitation wavelengths λ i , to obtain an emission map of said at least one tissue. 
     
     
       4. An optical imaging method according to  claim 1  or  2 , wherein the method further comprises using an optical imaging device operating in transmission mode and including a source of incident radiations and a detector which are situated on respective both opposite sides of said at least one tissue. 
     
     
       5. An optical imaging method according to  claim 1  or  2 , wherein the method further comprises using an optical imaging device including a source of incident radiations and a detector both of which are situated on a same side of said at least one tissue. 
     
     
       6. An optical imaging method according to  claim 5 , wherein the step b) further comprises deducing a backscattering map of the at least one tissue at the n different incident excitation wavelengths λ 1 . 
     
     
       7. An optical imaging method according to  claim 1  or  2 , wherein the n different incident excitation wavelengths λ i  are offset in pairs by an interval of at least 100 nm. 
     
     
       8. An optical imaging method according to  claim 1  or  2 , wherein the m maximum wavelengths λ′ j  of said emission bands B j  are offset in pairs by an interval of at least 100 nm. 
     
     
       9. An imaging method according to  claim 1  or  2 , wherein the n different incident excitation wavelengths λ i  are all of between 750 nm and 1000 nm. 
     
     
       10. An imaging method according to  claim 9 , wherein the m maximum wavelengths λ′ j  of said emission bands B j  are all of between 450 nm and 800 nm. 
     
     
       11. An optical imaging method according to  claim 1  or  2 , wherein the step b) comprises:
 successively exciting said at least one marker at two different excitation wavelengths λ 1  and λ 2 , said at least one marker being adapted to be excited by these two wavelengths λ 1  and λ 2  and to emit in response substantially same series S 1 , S 2  of two emission bands B 1  and B 2  having respective different maximum wavelengths λ′ 1  and λ′ 2  (n=m=2); and 
 detecting the two series S 1  and S 2  each comprising the two bands B 1  and B 2  emitted simultaneously. 
 
     
     
       12. An imaging method according to  claim 1  or  2 , wherein the at least one marker comprises a fluorophore based on at least one up-converting semiconductor inorganic nanocrystal. 
     
     
       13. An imaging method according to  claim 12 , wherein said nanocrystal includes at least:
 an oxide or an oxysulfide of a metal selected from a group consisting of yttrium, vanadium and rare earth elements; and 
 an emitter ion, which is a rare earth cation. 
 
     
     
       14. An imaging method according to  claim 1 , wherein said at least one marker further comprises an element chosen from a group consisting of chelates of gadolinium, nanoparticles of oxides of iron and nanoparticles of gadolinium, which element makes said at least one marker act as a contrast agent usable in magnetic resonance imaging, positron emission tomography, gammatomography or X-ray imaging. 
     
     
       15. An imaging method according to  claim 13 , wherein said nanocrystal is based on an yttrium oxide having a formula Y 2 O 3 : Er 3+ , Yb 3+ , where Er and Yb are respectively erbium and ytterbium and each is present in said nanocrystal at a doping rate from 1% to 20%. 
     
     
       16. An imaging method according to  claim 1  or  2 , wherein said at least one tissue is in vivo, said at least one marker is based on a fluorophore and biological ligand conjugate, which is an up-converting semiconductor nanocrystal and biomolecule conjugate. 
     
     
       17. An imaging method according to  claim 1  or  2 , wherein the step c) includes generating at least one of an emission map, absorption map, and backscattering map to define at least one region of interest of said at least one tissue to be analyzed by tomography. 
     
     
       18. An imaging method according to  claim 17 , further comprising using said at least one region of interest of said at least one tissue as a starting point for image reconstruction by tomography. 
     
     
       19. An imaging method according to  claim 17 , wherein said at least one of the emission map, absorption map, and backscattering map relates to an entire animal body.